Objective: To clarify the relationship between serum total testosterone and nocturia in males without an enlarged prostate. Design and Method: Among the 1029 male patients who visited our clinic for health screening from January 2010 to July 2014, 596 patients without benign prostate enlargement (BPE) were analyzed. To evaluate the effect of serum total testosterone on prevalence of nocturia and number of nocturia episodes, multivariate analyses were performed including the covariates of age, International Prostate Symptom Score (IPSS), International Index of Erectile Function (IIEF) score, body mass index, prostate specific antigen, prostate volume and maximal urinary flow rate. Results: Mean prostate volume was 21.70±4.34 ml and prevalence of nocturia (over 1 times/night) was 22.1%. Multivariate logistic regression analysis revealed positive associations of age (OR 1.048, P = 0.005), total IPSS (OR 1.217, P <0.001) and testosterone level (OR 1.115, P = 0.041) with the prevalence of nocturia. Although the mean testosterone level was progressively elevated as the number of nocturia episodes increased, no consistent association with the number of nocturia episodes was seen for testosterone after adjusting for age, IPSS, IIEF score and maximal urine flow rate. Conclusions: Serum total testosterone level is significantly positively associated with the prevalence of nocturia. Therefore, in men without enlarged prostate, testosterone seems to have an opposing role in the etiology of nocturia.